| Objective:1)Discuss whether h AMSCs possesses the characteristic of mesenchymal stem cells.2)To compare the morphology and proliferation of cells subjected to collagenase digestion and in situ culture of tissue pieces and to investigate the effect of b FGF on morphology,proliferation,relative m RNA level and protein expression in h ACLFs.3)To investigate the differentiation of h AMSCs into h ACLFs in vitro by using single induction,monolayer and Transwell co-culture systems with or without b FGFand TGF-β1.Methods: 1)The h AMSCs were separated through trypsin-enzyme digestion from the placenta of delivery women.The phenotypic characteristics of h AMSCs was tested through flow cytometry,the profileration ability was recorded by CCK-8 assay.Immunofluorescence staining were performed to investigate the expression of CK-19 and vimentin.The alizarin red,toluidine blue and oil red O staining were used to evaluate the differentiation effect of the h AMSCs into osteocytes,chondrocytes and lipocytes like.The ALP vitality assay were used to test the effect of the h AMSCs into the osteocytes.The real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR)analysis were performed to evaluate the relative genes expression of osteocytes,chondrocytes and lipocytes at day 7 and 21.2)Compare collagenase digestion and in situ tissue isolation and culture.An inverted phase contrast microscope and CCK-8(Cell Counting Kit-8)assay were used to detect morphology and proliferation,respectively.The phenotypic characteristics of the h ACLFs were identified by flow cytometry(FCM).After used the b FGF on h ACLFs at third passage,immunofluorescence staining were performed to investigate the expression of collagen type I and III,Fibronectin and Tenascin-C.The q RT-PCR and Western blot assays were performed to evaluate the relative gene including and protein expression in h ACLFs cultured with b FGF.3)The application of TGF-β1 associate b FGF as the induction agent were putted to LG-DMEM/F12 medium.The h AMSCs and h ACLFs were cultured to the 3rd passage,according the single induction,monolayer co-culture and Transwell co-culture system,our experiment were divided into six groups: h AMSCs were cultured with b FGF and TGF-β1(Group A);h AMSCs were cultured without b FGF and TGF-β1(Group B);monolayer h AMSCs and h ACLFs were co-cultured with both b FGF and TGF-β1(Group C);h AMSCs were cultured without b FGF and TGF-β1(Group D);h AMSCs and h ACLFs were co-cultured in a Transwell system with b FGF and TGF-β1(Group E);h AMSCs and h ACLFs were co-cultured in a Transwell system without b FGF and TGF-β1(Group F).The density of the two type of cells were adjusted into 104/ml and the ratio is 1:1.Before using monolayer co-culutre system,the h ACLFs in Group C and D were dealt with Arab-C(concentration: 5μg/ml)for 24 hours which distinctively killed the fibroblasts on synthetic phase.The morphology of the cells in each group were observed by inverted phase-contrast microscopy.The proliferation of the cells in each group was recorded by CCK-8 assay.The induction time of h AMSCs with b FGF and TGF-β1 in the different induction and co-culture systems was set at 7,14,and 21 days.The relative m RNA expression levels of the h ACLFs were measured by quantitative real-time polymerase chain reaction;the protein expression of the cells in each group were detected by immunofluorescence staining and Western blot,and collagen expression was observed by picrosirius red staining.Results: 1)A huge amount of hAMSCs which could firmly proliferate were obtained through trypsin-enzyme digestion.The flow cytometry result indicates that h AMSCs expresses mesenchymal stem cell phenotype.The h AMSCs on the third passage highly expressed the vimentin and have negative expression of CK-19.The Alizarin Red staining results showed of multiple mineralized nodules after osteocytes induction.The vitality of alkaline phosphatase(ALP)after induced at day 21 was significantly higher than induced at day 7(P<0.05).The Toluidine Blue staining showed positive results after chondrocytes induction.After adipogenic differentiation,strong refraction of lipid droplets were observed in cytoplasm of the cells.The Oil red O staining showed positive results.The PCR results showed the m RNA of relative genes expression of osteogenic,chondrogenic and adipogenic induction at day 21 upregulated significantly compared with the results at day 7(P < 0.05)2)The flow cytometry result indicates that isolated hACLFs expresses fibroblasts phenotype.The morphology of the h ACLFs isolated by collagenase digestion and in situ culture of tissue pieces was different from the primary to the 2nd passage.The few cells obtained by the collagenase isolation method were characterized as short rods with an irregular shape,whereas the cells isolated from tissue pieces in situ appeared to have a long spindle shape and bipolar-like growth.The proliferation of the cells obtained by in situ culture was higher than that of the cells obtained by collagenase digestion(P<0.05).The h ACLFs at the 3rd passage that were cultured with b FGF exhibited a better proliferation rate than that of the cells cultured without b FGF(P<0.05).The morphology of h ACLFs at the 3rd passage that were cultured with b FGF displayed a spindle shape,fibroblast-like growth and obvious polarity.Immunofluorescence staining showed a significant increase in the expression of collagen type I and III,fibronectin and tenascin-C in h ACLFs cultured with b FGF.Additionally,the expression of the collagen type I and III,fibronectin,tenascin-C,matrix metalloproteinase-2,and lysyl oxidase-1 m RNAs was upregulated in the h ACLFs cultured with b FGF(P<0.05).According to Western blot analysis,the levels of collagen type I and III in h ACLFs cultured with b FGF were significantly higher than those in h ACLFs cultured without b FGF(P<0.05).3)In the groups cultured with b FGF and TGF-β1,cell proliferation significantly increased compared with the non-induced groups(P<0.05).The protein expression of collagen types I and III,fibronectin,and tenascin-C increased over time,and the expression levels were higher in the induced groups than in the non-induced groups and the protein expression in induced Transwell co-culture group were higher than other groups at day 21.The m RNA levels of collagen types I and III,fibronectin,tenascin-C,matrix metalloproteinase 2,lysyl oxidase 1,and α-smooth muscle actin were upregulated on days 14 and 21(P<0.05)and the m RNA exression of induced Transwell co-culture group at day 21 were significantly upregulated.The Western blot results showed the collagen type I and III expression of induced Transwell co-culture group at day 21 was significantly higher than other groups(P<0.05).The picrosirius red staining results also demonstrated the total collagen expression of induced Transwell co-culture group at day 21 was significantly higher than other groups which the difference have statistical meaning(P<0.05).Conclusion: 1)The hAMSCs have the basic characteristics of MSCs and possess good proliferation ability.There were good effects of differentiation into osteocytes,chondrocytes and lipocytes with a stable induced environment in vitro in a fixed environment,which indicated h AMSCs have the potential in inducing into osteoblasts,chondrocytes and adipocytes and h AMSCs could selected as the seed cells for tissue engineering.2)The h ACLFs were successfully obtained and the h ACLFs possessed the phenotypic characteristics of fibroblasts.In situ isolation of tissue pieces enhanced the proliferation of h ACLFs in vitro,also,obtained considerable number of cells which the shape were more tend to the fibroblasts.3)The h ACLFs cultured with b FGF exhibited increased proliferation and expression of genes and proteins that increase the bioactivity of the h ACLFs.4)Using b FGF associate TGFβ-1 could enhanced the differentiation effect of h AMSCs into h ACLFs;compared with the single induction,the monolayer co-culture and Transwell co-culture system could be a better choice in differentiation process.But Trasnswell co-culture system have a strong and greate effect as to the m RNA level,protein and collagen expression which is superior to monolayer co-culture system.The Transwell co-culture system could imitate the h ACLFs growth environment in vivo which the induced h AMSCs are extremely close to the normal h ACLFs,in addition,combined with b FGF and TGFβ-1 will further improve the effect of differentiation of h AMSCs into h ACLFs.Thus,and may be a superior choice for h ACLF differentiation via Transwell co-culture systems. |
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